APPLICATION NOTE

WITec GmbH, Lise-Meitner-Str. 6, 89081 Ulm, Germany
phone +49 (0) 731 140 700, fax +49 (0) 731 140 70 200
info@WITec.de, www.WITec.de

Correlative Raman Imagingof P0lymeric Materials

Confocal Raman microscopy is a ​promising technique for highresolution, non-destructive andlabel-free analysis of polymers intwo or three dimensions. It can easily
be combined with other imaging
techniques such as AFM or SNOM.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

The Raman principle

The Raman effect is based on the inelastic
scattering of light by the molecules ofgaseous, liquid or solid materials. Theinteraction of a molecule with photons
causes vibrations of its chemical bonds,
leading to specific energy shifts in thescattered light. Thus, any given chemicalcompound produces a particular Raman
spectrum when excited and can be easily
identified by this individual “fingerprint.”
Raman spectroscopy is a well-established,label-free and non-destructive method foranalyzing the molecular composition of asample.

No need for compromises

Raman imaging

In Raman imaging, a confocal microscopeis combined with a spectrometer anda Raman spectrum is recorded at every
image pixel. The resulting Raman image
visualizes the distribution of the sample’scompounds. Due to the high confocality of
WITec Raman systems, volume scans and3D images can also be generated.

The Raman effect is extremely weak, so
every Raman photon is important forimaging. Therefore WITec Raman imaging
systems combine an exceptionallysensitive confocal microscope with anultra-high throughput spectrometer(UHTS). Precise adjustment of all opticaland mechanical elements guarantees thehighest resolution, outstanding speed andextraordinary sensitivity - simultaneously!
This optimization allows the detectionof Raman signals of even weak Raman
scatterers and extremely low material
concentrations or volumes with thelowest excitation energy levels. This isan unrivaled advantage of WITec systems.

Resolution

Lateral resolution is physicallylimited to ~200 nm, dependingon the wavelength of theincident light.

Speed

The more sensitive a system is, the shorterthe acquisition time for a single spectrum.
WITec’s Ultrafast Raman Imaging reduces
acquisition times for single Raman spectra
down to well below 1 ms.

Sensitivity

A high confocality increases the signal-to-noise ratio by reducing
the background. With the UHTS Series, WITec developed lens-based,
wavelength-optimized spectrometers with a spectral resolution
down to 0.1 cm-1 relative wavenumbers.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.deCorrelative high-resolution Raman-AFM-SNOM imaging of a three-polymer mixture
A three-component polymer blend wasstudied by using a combination of Atomic
Force Microscopy (AFM), Scanning Nearfield Microscopy (SNOM) and confocal
Raman imaging.
While AFM operating in AC Mode recor­
ded topographic information and local
mechanical characteristics, SNOM
detected optical properties with resolution
far below the diffraction limit andRaman imaging revealed the molecularcomposition of the sample. Correlated
AFM – SNOM – confocal Raman imaging,
provides deep insight into the chemicaland physical properties of polymer
mixtures on a sub-micrometer scale.
The phase separation of a thin film
blend of 1:1:1 polysterene (PS), styrenebutadiene-rubber (SBR) and ethyl-hexyl
acrylate (EHA) was analyzed with a WITecalpha300 RAS microscope featuring all
three analysis methods integrated within
one instrument. The sample, spin-coatedonto glass, remained in place throughout
all measurements, allowing for images tobe correlated. Evalua­tion of the acquired
data and generation of depth profiles
was accomplished with the WITec Controlsoftware.
The topography of the sample measuredwith AFM reveals a three-level structure(Figure 1A). The simultaneously recorded
phase image (Figure 1B) shows a fine
netlike texture at the lowest topographiclevel, material containing small spheres in
the intermediate layer and an amorphous
pattern in the uppermost substance. The
SNOM image acquired from the same
sample section indicates that the thinnest
areas of the samples are opaque (Figure 1C). These areas contain EHA only, as canbe seen in the Raman image (Figure 1E)

ABCDEF

Figure 1: Correlative, high-resolution AFM – SNOM – confocal Raman imaging of a 1:1:1 mixture of PS,
SBR and EHA.
(A, B) Using AFM in tapping mode the sample’s topography (A) and its phase image (B) were measured simultaneously. (C) With SNOM the transparency of the same area was determined with the dark
spots being opaque. (D, E) Concurrently with the AFM and SNOM measurements the Raman spectra(D) were recorded for each pixel of the area and displayed as a false color image (E). PS (red), EHA(green), SBR (violet), mixed spectrum of SBR and EHA (blue). (F) Overlay of the AFM phase and Ramanimages.

that was generated from the Ramanspectra (Figure 1D). The uppermost features of the sample appear to be PS, which
is known to form spheres. The perfectcorrelation of topography, phase separation, SNOM and molecular composition isillustrated by the overlay of the AFM phaseand Raman images (Figure 1F).
All correlated structural, physical andchemical data lead to the conclusion that
the EHA forms the lowermost layer on themicroscope slide. It is covered by a layer of

SBR as indicated by the blended (EHA-SBR)
Raman spectrum. PS spheres are submerged in and protrude out of this double-layer polymer film.
The combination of AFM, SNOM and confocal Raman imaging in a single instrument enables easy-to-use, non-destructive,
physical and chemical characterizationof heterogeneous materials at very highresolution.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

Characterization of polymer blends with correlative microscopy:
Confocal Raman imaging and Atomic Force Microscopy
Polymers play an essential role in modernmaterials science. Due to their widely
varying mechanical and chemical properties they are used in almost every fieldof application and remain important fordeveloping new materials with demanding requirements. Thus, knowledge of themorphology and chemical composition ofheterogeneous polymeric materials on a
sub-micrometer scale is crucial.
Certain properties, however, are difficult to
study with conventional characterization
techniques, which are often not able tochemically differentiate materials with
sufficient spatial resolution and without
damaging, staining or otherwise treating
them.

Correlative microscopy comprised of
Atomic Force Microscopy (AFM) and confocal Raman imaging opens new avenues
for the analysis of advanced polymericmaterials when a detailed understandingof physical and chemical properties isrequired. While AFM records topographic information and local mechanicalcharacteristics along and perpendicular tothe surface of a sample with high spatial
resolution, Raman imaging reveals its
molecular composition.
The samples remained in place during
all measurements, allowing the resulting
images to be correlated. The WITec Projectsoftware enables a comprehensive evaluation of the acquired data and the generation of depth profiles.

Figure 2: AFM image of a PMMA-SBR blend. The scan area was 20 x 20 x 0.03 µm3..

Thin films of a mixture of two polymers
were studied. Polymethylacrylate (PMMA)and styrene-butadiene-rubber (SBR) weremixed and spin coated onto a glass coverslip. A WITec alpha300 RA microscope wasused for imaging.
The mixture was analyzed with AFM in
the AC Mode (Figure 2). The topographic
image reveals spherical structures which
protrude from the surrounding material.
Their chemical nature was determined bytheir Raman spectra.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.deAB

Figure 3: Raman image of a PMMA-SBR blend.
(A) Raman spectra of PMMA (red) and SBR (blue). PMMA was identified by its typical C=O stretching band and SBR molecules by
their C=C double bond stretching band. (B) Confocal Raman image. PMMA (red) forms round structures in the surrounding SBR(blue).

The Raman spectra of the molecules inthese island-like structures contain aband at 1735 cm-1 relative wavenumbers(Figure 3A, red). This is associated with C=O
double bond stretching, identifying thesubstance as PMMA. The area between thespheres consists of SBR. It was identified

by a Raman band at 1640 cm-1 relative
wavenumbers that is characteristic for theC=C double bonds of the SBR molecules(Figure 3A, blue). The distribution of thepolymer was visualized by Raman imaging(Figure 3B).

ATo determine the thickness of the polymer
blend, the film was scratched with a razor
blade along the black line indicated in theAFM image (Figure 4A). The depth profile
recorded along this line indicates that thePMMA islands are up to 70 nm high andprotrude from the SBR film that is 35 nm
high (Figure 4B).

B

Figure 4: AFM image and depth profile of a PMMA-SBR mixture.

(A) AFM image of the PMMA-SBR blend spin-coated on glass. The film was scratched with a sharp razor along the black line anda depth-profile was measured. (B) Depth profile along the black line in(A).
Image parameters: scan range 10 x 12 x 0.6 µm3..

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

Confocal Raman imaging for depth profiling of polymer films and coatings
A full analysis of a heterogenous polymer
blend not only includes the two-dimensional arrangement of its individualcomponents but also their distribution
in the third dimension. A comprehensive,
three-dimensional characterization of polymers such as films and coatings can beachieved with confocal Raman microscopy. Films and coatings play an importantrole in many fields of application such as
food packaging, drug delivery and medicaldevices or adhesives.
While the resolution of two-dimensional
Raman imaging is limited by diffraction,
the quality of depth resolution relies on
the confocality of the microscope. WITec
confocal Raman microscopes combine ahighly sensitive confocal microscope anda high-transmission Raman spectroscopy
system. The WITec Control software canextract a variety of properties from asingle measurement such as peak width,
center of mass or peak position of certain
Raman lines. For depth profiling measurements, the focal plane can be moved in
the z-direction when performing either
x-z scans or generating x-y image stacks
in the z-direction. Here, the microscope‘sAAB

Figure 5: Raman imaging of theinner coating of an orange juicecontainer.
(A) Recorded Raman spectra. (B)Corresponding Raman image.

capabilities are demonstrated by imaging
the inner polymer coating of an orange
juice container and of polymer layers onpaper.
The inner plastic film of a paper beveragecontainer was investigated by performing
an x-z scan. Within the acquired multispectrum file, four distinct spectra wereobserved, each representing a specificchemical compound within the beveragecontainer coating (Figure 5A). The Raman
image generated from the spectra showsthat the coating is about 80 μm thickand consists of five layers of different
thicknesses made up of four compounds(Figure 5B).

B

In another experiment, confocal Raman
depth scans provided insight into thelayered structure of polymers that wereapplied to the surface of paper in order tomake it adhesive.

Figure 6: Raman imaging of adhesive polymeric layers on a paper substrate.
(A) Recorded Raman spectra. (B) Raman depth profile. Image parameters: 50 x 40 µm2, 150 x 50 pixels(= 7500 spectra), 0.1 sec/spectrum acquisition time. The two different polymers (red and green) form
separate layers on the paper (blue, yellow).

The two polymers and the paper (consisting of cellulose) were identified by their
Raman spectra (Figure 6A). The Raman
depth scan (Figure 6B) shows that bothpolymers are immiscible and form a sharp
interface with each other and with thepaper beneath.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, GermanyTel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

Correlative AFM/Raman Microscopy of bioinspired nanofibers
Natural fibers display many intriguing
qualities such as extreme tearing strength,
high flexibility and elasticity, self-crimping
and varying chemical properties or morphologies that bio-engineers would like tocopy for the design of synthetic materials.
Several methods have been developed
to produce artificial fibers, one of which
is electrospinning. This technique uses astrong electric field to overcome surfacetension to produce a spinning jet fromsolutions or melts. Thus, electrospinning
results in continuous artificial nanofibers
with diameters down to the nanometer
range. The fibers can be made of one ormore materials.
The experimental conditions and solventsused have a great impact on the morphology of the resulting fibers, which is
why the products need to be investigatedthoroughly.
Here, we show a correlative analysis of
atomic force microscopy and confocal
Raman imaging of two-component fibers.
They were produced by scientists at theBayreuth Center for Colloids and Interfacesat Bayreuth University (Germany) and consist of silk from the silkworm Bombyx moriand polylactic acid. The fibers were spunfrom side-by-side nozzles as described byPeng et al. (1).
Microscopy was performed with a WITecalpha300 RA confocal Raman microscope.
This instrument is equipped with AFM
and Raman imaging capabilities, so thatthe sample does not need to be moved
between the measurements. AFM wasconducted in AC mode with Pulsed Force
Mode. Raman imaging was performedwith a 100x/NA 0.9 objective, 532 nm
excitation laser, and a WITec UHTS300
spectrometer equipped with a 600 lines/mm grating. Data analysis was carried out
with the WITec Suite FIVE software.
The topography of two nanofibers wasinvestigated with the high-resolution AFMABCDsilkEpolylactic acid

Figure 7: Correlative AFM - Confocal Raman imaging of fibers(A) Topography of two fibers with height profile (B) at the red and blue positions marked in (A). (C) Raman spectra of silk and polylactic acid. Green = silk, blue = polylactic acid. (D) Confocal Raman imageof the fiber. Note that at a right angle to the two-component fiber lies a smaller fiber consisting ofpolylactic acid only. (E) Depth scan along the z-axis of the two-component fiber.

capability of the microscope (Figure 7A).
Two cross section measurements wereperformed as indicated. One fiber wasroughly 900 nm thick whereas the otherwas only about 100 nm thick (Figure 7B).
The chemical nature of each fiber wasdetermined by Raman analysis. Silk andpolylactic acid could be easily distinguished by their Raman spectra (Figure 7C).
To generate the confocal Raman image(Figure 7D), complete Raman spectra
were acquired at each image pixel. The
color-coded image (silk in green, polylactic
acid in blue) shows the side-by-side distribution of the two components along theaxis of the thicker fiber. The second fiber
consists of polylactic acid only.
A depth scan along the z-axis of theside-by-side fiber was performed and underlines the aligned, parallel positioning ofthe fibers‘ components (Figure 7E).

This data and other examples (2) illustratethat high-resolution AFM and Raman imaging is a superb combination for the precise morphological and chemical characterization of two-component nanofibers.

1.
2.

Peng et al., Macromolecular Materials andEngineering 301, 48-55, 2016
Gernhardt et al., Macromolecular Materials and Engineering 303, 1700248, 2017

WITec Microscope Seriesalpha300 S: Scanning
Near-field Optical Microscopealpha300 A:
Atomic Force Microscopealpha300 R:
Confocal Raman Microscopealpha300 apyron: AutomatedConfocal Raman Microscopealpha300 Ri: Inverted
Confocal Raman Microscope

RISE: Raman Imaging –Scanning Electron Microscopealpha300 access: Confocal
Micro-Raman System

https://raman.oxinst.com
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